Chemokine Binding to Tenascin-C Influences Chemokine-Induced Immune Cell Migration.

Tenascin-C (TNC) is a complex glycoprotein of the extracellular matrix (ECM) involved in a plethora of (patho-)physiological processes, such as oncogenesis and inflammation. Since chemokines play an essential role in both disease processes, we have investigated here the binding of TNC to some of the key chemokines, namely CCL2, CCL26, CXCL8, CXCL10, and CXCL12. Thereby, a differential chemokine-TNC binding pattern was observed, with CCL26 exhibiting the highest and CCL2 the lowest affinity for TNC. Heparan sulfate (HS), another member of the ECM, proved to be a similarly high-affinity ligand of TNC, with a Kd value of 730 nM. Chemokines use glycosa-minoglycans such as HS as co-receptors to induce immune cell migration. Therefore, we assumed an influence of TNC on immune cell chemotaxis due to co-localization within the ECM. CCL26- and CCL2-induced mobilization experiments of eosinophils and monocytes, respectively, were thus performed in the presence and the absence of TNC. Pre-incubation of the immune cells with TNC resulted in a 3.5-fold increase of CCL26-induced eosinophil chemotaxis, whereas a 1.3-fold de-crease in chemotaxis was observed when monocytes were pre-incubated with CCL2. As both chemokines have similar HS binding but different TNC binding affinities, we speculate that TNC acts as an attenuator in monocyte and as an amplifier in eosinophil mobilization by impeding CCL2 from binding to HS on the one hand, and by reinforcing CCL26 to bind to HS on the other hand.

Investigating Chemokine-Matrix Networks in Breast Cancer: Tenascin-C Sets the Tone for CCL2.

Bidirectional dialogue between cellular and non-cellular components of the tumor microenvironment (TME) drives cancer survival. In the extracellular space, combinations of matrix molecules and soluble mediators provide external cues that dictate the behavior of TME resident cells. Often studied in isolation, integrated cues from complex tissue microenvironments likely function more cohesively. Here, we study the interplay between the matrix molecule tenascin-C (TNC) and chemokine CCL2, both elevated in and associated with the progression of breast cancer and playing key roles in myeloid immune responses. We uncover a correlation between TNC/CCL2 tissue levels in HER2+ breast cancer and examine the physical and functional interactions of these molecules in a murine disease model with tunable TNC levels and in in vitro cellular and cell-free models. TNC supported sustained CCL2 synthesis, with chemokine binding to TNC via two distinct domains. TNC dominated the behavior of tumor-resident myeloid cells; CCL2 did not impact macrophage survival/activation whilst TNC facilitated an immune suppressive macrophage phenotype that was not dependent on or altered by CCL2 co-expression. Together, these data map new binding partners within the TME and demonstrate that whilst the matrix exerts transcriptional control over the chemokine, each plays a distinct role in subverting anti-tumoral immunity.

The Role of Heparan Sulfate in CCL26-Induced Eosinophil Chemotaxis.

Proinflammatory chemokine ligand 26 (CCL26, eotaxin-3) mediates transendothelial cell migration of eosinophils by binding and activating the G-protein-coupled (GPC) chemokine receptor 3 on the surface of eosinophilic cells. Here we have investigated the role of glycosaminoglycans (GAGs) as potential co-receptors in the process of CCL26-induced eosinophil chemotaxis. For this purpose, we have first identified the GAG-binding site of CCL26 by a site-directed mutagenesis approach in the form of an alanine screening. A panel of GAG-binding-deficient mutants has been designed, generated, and analyzed with respect to their binding affinities to heparan sulphate (HS) by isothermal fluorescence titration studies. This showed that basic amino acids in the α-helical part of CCL26 are strongly involved in GAG-binding. In chemotaxis experiments, we found that decreased GAG-binding affinity correlated with decreased chemotactic activity, which indicates an involvement of GAGs in eosinophil migration. This was further proven by the negative impact of heparinase III treatment and, independently, by the incubation of eosinophils with an anti heparan sulfate antibody. We finally investigated eosinophils' proteoglycan (PG) expression patterns by real-time PCR, which revealed the highest expression level for serglycin. Including an anti-serglycin antibody in CCL26-induced eosinophil migration experiments reduced the chemotaxis of these immune cells, thereby proving the dependence of eosinophil mobilization on the proteoglycan serglycin.

Single domain shark VNAR antibodies neutralize SARS-CoV-2 infection in vitro.

Single domain shark variable domain of new antigen receptor (VNAR) antibodies can offer a viable alternative to conventional Ig-based monoclonal antibodies in treating COVID-19 disease during the current pandemic. Here we report the identification of neutralizing single domain VNAR antibodies selected against the severe acute respiratory syndrome coronavirus 2 spike protein derived from the Wuhan variant using phage display. We identified 56 unique binding clones that exhibited high affinity and specificity to the spike protein. Of those, 10 showed an ability to block both the spike protein receptor binding domain from the Wuhan variant and the N501Y mutant from interacting with recombinant angiotensin-converting enzyme 2 (ACE2) receptor in vitro. In addition, three antibody clones retained in vitro blocking activity when the E484K spike protein mutant was used. The inhibitory property of the VNAR antibodies was further confirmed for all 10 antibody clones using ACE2 expressing cells with spike protein from the Wuhan variant. The viral neutralizing potential of the VNAR clones was also confirmed for the 10 antibodies tested using live Wuhan variant virus in in vitro cell infectivity assays. Single domain VNAR antibodies, due to their low complexity, small size, unique epitope recognition, and formatting flexibility, should be a useful adjunct to existing antibody approaches to treat COVID-19.

Isolation and Characterization of Heparan Sulfate from Human Lung Tissues.

Glycosaminoglycans are a class of linear, highly negatively charged, O-linked polysaccharides that are involved in many (patho)physiological processes. In vitro experimental investigations of such processes typically involve porcine-derived heparan sulfate (HS). Structural information about human, particularly organ-specific heparan sulfate, and how it compares with HS from other organisms, is very limited. In this study, heparan sulfate was isolated from human lung tissues derived from five donors and was characterized for their overall size distribution and disaccharide composition. The expression profiles of proteoglycans and HS-modifying enzymes was quantified in order to identify the major core proteins for HS. In addition, the binding affinities of human HS to two chemokines-CXCL8 and CCL2-were investigated, which represent important inflammatory mediators in lung pathologies. Our data revealed that syndecans are the predominant proteoglycan class in human lungs and that the disaccharide composition varies among individuals according to sex, age, and health stage (one of the donor lungs was accidentally discovered to contain a solid tumor). The compositional difference of the five human lung HS preparations affected chemokine binding affinities to various degrees, indicating selective immune cell responses depending on the relative chemokine-glycan affinities. This represents important new insights that could be translated into novel therapeutic concepts for individually treating lung immunological disorders via HS targets.

Inhibition of Drug-Induced Liver Injury in Mice Using a Positively Charged Peptide That Binds DNA.

Hepatic cell death occurs in response to diverse stimuli such as chemical and physical damage. The exposure of intracellular contents such as DNA during necrosis induces a severe inflammatory response that has yet to be fully explored therapeutically. Here, we sought means to neutralize the ability of extracellular DNA to induce deleterious tissue inflammation when drug-induced liver injury had already ensued. DNA exposure and inflammation were investigated in vivo in drug-induced liver injury using intravital microscopy. The necrotic DNA debris was studied in murine livers in vivo and in DNA debris models in vitro by using a positively charged chemokine-derived peptide (MIG30; CXCL9[74-103]). Acetaminophen-induced liver necrosis was associated with massive DNA accumulation, production of CXC chemokines, and neutrophil activation inside the injured tissue. The MIG30 peptide bound the healthy liver vasculature and, to a much greater extent, to DNA-rich necrotic tissue. Moreover, MIG30 bound extracellular DNA directly in vivo in a charge-dependent manner and independently of glycosaminoglycans and chemokines. Post-treatment of mice with MIG30 reduced mortality, liver damage, and inflammation significantly. These effects were not observed with a control peptide that does not bind DNA. Mechanistically, MIG30 inhibited the interaction between DNA and histones, and promoted the dissociation of histones from necrotic debris. MIG30 also inhibited the pro-inflammatory effect of CpG DNA, as measured by a reduction in CXCL8 production, indicating that MIG30 disturbs the ability of DNA to induce hepatic inflammation. Conclusion: The use of DNA-binding peptides reduces necrotic liver injury and inflammation, even at late timepoints.

Development of Molecules Antagonizing Heparan Sulfate Proteoglycans.

Heparan sulfate proteoglycans (HSPGs) occur in almost every tissue of the human body and consist of a protein core, with covalently attached glycosaminoglycan polysaccharide chains. These glycosaminoglycans are characterized by their polyanionic nature, due to sulfate and carboxyl groups, which are distributed along the chain. These chains can be modified by different enzymes at varying positions, which leads to huge diversity of possible structures with the complexity further increased by varying chain lengths. According to their location, HSPGs are divided into different families, the membrane bound, the secreted extracellular matrix, and the secretory vesicle family. As members of the extracellular matrix, they take part in cell-cell communication processes on many levels and with different degrees of involvement. Of particular therapeutic interest is their role in cancer and inflammation as well as in infectious diseases. In this review, we give an overview of the current status of medical approaches to antagonize HSPG function in pathology.

Cytokines and Chemokines in SARS-CoV-2 Infections-Therapeutic Strategies Targeting Cytokine Storm.

The recently identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, the cause of coronavirus disease (COVID-19) and the associated ongoing pandemic, frequently leads to severe respiratory distress syndrome and pneumonia with fatal consequences. Although several factors of this infection and its consequences are not completely clear, the presence and involvement of specific chemokines is undoubtedly crucial for the development and progression of COVID-19. Cytokine storm and the often-resulting cytokine release syndrome (CRS) are pathophysiological hallmarks in COVID-19 infections related to its most severe and fatal cases. In this hyperinflammatory event, chemokines and other cytokines are highly upregulated and are therefore not fulfilling their beneficial function in the host response anymore but causing harmful effects. Here, we present the recent views on the involvement of chemokines and selected cytokines in COVID-19 and the therapeutics currently in clinical development targeting or interfering with them, discussing their potentials in the treatment of COVID-19 infections.

Enoxaparin and Pentosan Polysulfate Bind to the SARS-CoV-2 Spike Protein and Human ACE2 Receptor, Inhibiting Vero Cell Infection.

As with many other pathogens, SARS-CoV-2 cell infection is strongly dependent on the interaction of the virus-surface Spike protein with the glycosaminoglycans of target cells. The SARS-CoV-2 Spike glycoprotein was previously shown to interact with cell-surface-exposed heparan sulfate and heparin in vitro. With the aim of using Enoxaparin as a treatment for COVID-19 patients and as prophylaxis to prevent interpersonal viral transmission, we investigated GAG binding to the Spike full-length protein, as well as to its receptor binding domain (RBD) in solution by isothermal fluorescence titration. We found that Enoxaparin bound to both protein variants with similar affinities, compared to the natural GAG ligand heparan sulfate (with Kd-values in the range of 600-680 nM). Using size-defined Enoxaparin fragments, we discovered the optimum binding for dp6 or dp8 for the full-length Spike protein, whereas the RBD did not exhibit a significant chain-length-dependent affinity for heparin oligosaccharides. The soluble ACE2 receptor was found to interact with unfractionated GAGs in the low µM Kd range, but with size-defined heparins with clearly sub-µM Kd-values. Interestingly, the structural heparin analogue, pentosan polysulfate (PPS), exhibited high binding affinities to both Spike variants as well as to the ACE2 receptor. In viral infection experiments, Enoxaparin and PPS both showed a strong inhibition of infection in a concentration range of 50-500 µg/mL. Both compounds were found to retain their inhibitory effects at 500 µg/mL in a natural biomatrix-like human sputum. Our data suggest the early topical treatment of SARS-CoV-2 infections with inhaled Enoxaparin; some clinical studies in this direction are already ongoing, and they further imply an oral or nasal prophylactic inactivation of the virus by Enoxaparin or PPS for the prevention of inter-personal viral transmission.

Structural Fuzziness of the RNA-Organizing Protein SERF Determines a Toxic Gain-of-interaction.

The mechanisms by which protein complexes convert from functional to pathogenic are the subject of intensive research. Here, we report how functionally unfavorable protein interactions can be induced by structural fuzziness, i.e., by persisting conformational disorder in protein complexes. We show that extreme disorder in the bound state transforms the intrinsically disordered protein SERF1a from an RNA-organizing factor into a pathogenic enhancer of alpha-synuclein (aSyn) amyloid toxicity. We demonstrate that SERF1a promotes the incorporation of RNA into nucleoli and liquid-like artificial RNA-organelles by retaining an unusually high degree of conformational disorder in the RNA-bound state. However, this type of structural fuzziness also determines an undifferentiated interaction with aSyn. RNA and aSyn both bind to one identical, positively charged site of SERF1a by an analogous electrostatic binding mode, with similar binding affinities, and without any observable disorder-to-order transition. The absence of primary or secondary structure discriminants results in SERF1a being unable to select between nucleic acid and amyloidogenic protein, leading the pro-amyloid aSyn:SERF1a interaction to prevail in the cytosol under conditions of cellular stress. We suggest that fuzzy disorder in SERF1a complexes accounts for an adverse gain-of-interaction which favors toxic binding to aSyn at the expense of nontoxic RNA binding, thereby leading to a functionally distorted and pathogenic process. Thus, structural fuzziness constitutes a direct link between extreme conformational flexibility, amyloid aggregation, and the malfunctioning of RNA-associated cellular processes, three signatures of neurodegenerative proteinopathies.

Increased Aggregation Tendency of Alpha-Synuclein in a Fully Disordered Protein Complex.

The recent discovery of biologically active fully disordered, so called random fuzzy protein-protein interactions leads to the question of how the high flexibility of these protein complexes correlates to aggregation and pathologic misfolding. We identify the structural mechanism by which a random fuzzy protein complex composed of the intrinsically disordered proteins alpha-Synuclein and SERF1a is able to potentiate cytotoxic aggregation. A structural model derived from an integrated NMR/SAXS analysis of the reconstituted aSyn:SERF1a complex enabled us to observe the partial deprotection of one precise aSyn amyloid nucleation element in the fully unstructured ensemble. This minimal exposure was sufficient to increase the amyloidogenic tendency of SERF1a-bound aSyn. Our findings provide a structural explanation of the previously observed pro-amyloid activity of SERF1a. They further demonstrate that random fuzziness can trigger a structurally organized disease-associated reaction such as amyloid polymerization.

Molecular dynamics simulations of the chemokine CCL2 in complex with pull down-derived heparan sulfate hexasaccharides.

BACKGROUND: Binding of chemokines to glycosaminoglycans (GAGs) is a crucial step in leukocyte recruitment to inflamed tissues. METHODS: A disaccharide compositional analysis of the HS dp6 fraction in combination with MS analysis of the CCL2-depleted dp6 fraction was the basis for target GAG ligand structure suggestions. Four experimentally-derived heparan sulfate hexasaccharides, two potentially chemokine-specific and two unspecific, have been docked to CCL2. Subsequent 300 ns molecular dynamics simulations were used to improve the docked complexes. RESULTS: Hexasaccharides with four sulfations and no acetylations are suggested for selective and high affinity chemokine binding. Using the Antithromin-III/heparin complex as positive control for docking, we were able to recover the correct complex structure only if the previously liganded ATIII structure was used as input. Since the liganded structure is not known for a CCL2-GAG complex, we investigated if molecular dynamics simulations could improve initial docking results. We found that all four GAG oligosaccharides ended up in close contact with the known binding residues after about 100 ns simulation time. CONCLUSIONS: A discrimination of specific vs. unspecific CCL2 GAG ligands is not possible by this approach. Long-time molecular dynamics simulations are, however, well suited to capture the delicate enthalpy/entropy balance of GAG binding and improve results obtained from docking. GENERAL SIGNIFICANCE: With the comparison of two methods, MS-based ligand identification and molecular modelling, we have shown the current limitations of our molecular understanding of complex ligand binding which is could be due to the numerical inaccessibility of ligand-induced protein conformational changes.

Therapeutic strategies to target microbial protein-glycosaminoglycan interactions.

Glycans are involved in a plethora of human pathologies including infectious diseases. Especially, glycosaminoglycans (GAGs), like heparan sulfate and chondroitin sulfate, have been found to be involved in different crucial stages of microbial invasion. Here, we review various therapeutic approaches, which target the interface of host GAGs and microbial proteins and discuss their limitations and challenges for drug development.

Anti-inflammatory effects of the GAG-binding CXCL9(74-103) peptide in dinitrofluorobenzene-induced contact hypersensitivity in mice.

BACKGROUND: To recruit leucocytes to an inflammatory site, chemokine binding to glycosaminoglycans (GAGs) is critical. Therefore, strategies to interfere with this interaction, aiming at the production of anti-inflammatory agents, were developed. These include production of modified chemokines without affinity for G protein-coupled receptors but with enhanced affinity for GAGs. Such modified chemokines compete with functional chemokines for GAG binding, prevent chemokine immobilization and presentation, and inhibit leucocyte migration. In addition to modified chemokines, a GAG-binding peptide consisting of the 30 COOH-terminal residues of CXCL9, that is CXCL9(74-103), inhibited CXCL8- and monosodium urate crystal-induced neutrophil migration. OBJECTIVE: We wanted to explore whether interference with chemokine-GAG interactions by CXCL9(74-103) reduces inflammation in neutrophil-dependent dinitrofluorobenzene-induced contact hypersensitivity. METHODS: For this study, we evaluated several inflammatory parameters, including ear swelling and the levels of chemokines, cytokines, proteases and neutrophils in the ears of dinitrofluorobenzene-induced mice treated with CXCL9(74-103) or buffer. RESULTS: One intravenous injection of CXCL9(74-103), just before painting with dinitrofluorobenzene on the ear, did not affect protein levels of the major murine neutrophil attractant, that is CXCL6, in this contact hypersensitivity model. However, IL-6, CXCL1, CCL2 and matrix metalloproteinase-9 (MMP-9) protein concentrations and peroxidase activity in challenged ears were reduced. In addition, intravenous injection of the CXCL9-derived peptide led to a reduced ear swelling response, indicating that the locally produced chemokines were hindered to attract leucocytes. The inhibiting potential of CXCL9(74-103) was explained by its competition for GAG binding with CXCL1, CXCL6 and CCL3 and inhibition of transendothelial migration of neutrophils to CXCL6. CONCLUSIONS AND CLINICAL RELEVANCE: The CXCL9(74-103) peptide inhibited dinitrofluorobenzene-induced infiltration of neutrophils and neutrophil-dependent inflammation in ears. Therefore, CXCL9(74-103) may be a lead molecule for the development of therapeutic peptides or peptide derivatives that compete with functional chemokines for GAG binding.

Novel Anti-Inflammatory Peptides Based on Chemokine-Glycosaminoglycan Interactions Reduce Leukocyte Migration and Disease Severity in a Model of Rheumatoid Arthritis.

Inflammation is characterized by the infiltration of leukocytes from the circulation and into the inflamed area. Leukocytes are guided throughout this process by chemokines. These are basic proteins that interact with leukocytes to initiate their activation and extravasation via chemokine receptors. This is enabled through chemokine immobilization by glycosaminoglycans (GAGs) at the luminal endothelial surface of blood vessels. A specific stretch of basic amino acids on the chemokine, often at the C terminus, interacts with the negatively charged GAGs, which is considered an essential interaction for the chemokine function. Short-chain peptides based on this GAG-binding region of the chemokines CCL5, CXCL8, and CXCL12γ were synthesized using standard Fmoc chemistry. These peptides were found to bind to GAGs with high affinity, which translated into a reduction of leukocyte migration across a cultured human endothelial monolayer in response to chemokines. The leukocyte migration was inhibited upon removal of heparan sulfate from the endothelial surface and was found to reduce the ability of the chemokine and peptide to bind to endothelial cells in binding assays and to human rheumatoid arthritis tissue. The data suggest that the peptide competes with the wild-type chemokine for binding to GAGs such as HS and thereby reduces chemokine presentation and subsequent leukocyte migration. Furthermore, the lead peptide based on CXCL8 could reduce the disease severity and serum levels of the proinflammatory cytokine TNF-α in a murine Ag-induced arthritis model. Taken together, evidence is provided for interfering with the chemokine-GAG interaction as a relevant therapeutic approach.

CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions.

Several acute and chronic inflammatory diseases are driven by accumulation of activated leukocytes due to enhanced chemokine expression. In addition to specific G protein-coupled receptor-dependent signaling, chemokine-glycosaminoglycan (GAG) interactions are important for chemokine activity in vivo. Therefore, the GAG-chemokine interaction has been explored as target for inhibition of chemokine activity. It was demonstrated that CXCL9(74-103) binds with high affinity to GAGs, competed with active chemokines for GAG binding and thereby inhibited CXCL8- and monosodium urate (MSU) crystal-induced neutrophil migration to joints. To evaluate the affinity and specificity of the COOH-terminal part of CXCL9 toward different GAGs in detail, we chemically synthesized several COOH-terminal CXCL9 peptides including the shorter CXCL9(74-93). Compared to CXCL9(74-103), CXCL9(74-93) showed equally high affinity for heparin and heparan sulfate (HS), but lower affinity for binding to chondroitin sulfate (CS) and cellular GAGs. Correspondingly, both peptides competed with equal efficiency for CXCL8 binding to heparin and HS but not to cellular GAGs. In addition, differences in anti-inflammatory activity between both peptides were detected in vivo. CXCL8-induced neutrophil migration to the peritoneal cavity and to the knee joint were inhibited with similar potency by intravenous or intraperitoneal injection of CXCL9(74-103) or CXCL9(74-93), but not by CXCL9(86-103). In contrast, neutrophil extravasation in the MSU crystal-induced gout model, in which multiple chemoattractants are induced, was not affected by CXCL9(74-93). This could be explained by (1) the lower affinity of CXCL9(74-93) for CS, the most abundant GAG in joints, and (2) by reduced competition with GAG binding of CXCL1, the most abundant ELR+ CXC chemokine in this gout model. Mechanistically we showed by intravital microscopy that fluorescent CXCL9(74-103) coats the vessel wall in vivo and that CXCL9(74-103) inhibits CXCL8-induced adhesion of neutrophils to the vessel wall in the murine cremaster muscle model. Thus, both affinity and specificity of chemokines and the peptides for different GAGs and the presence of specific GAGs in different tissues will determine whether competition can occur. In summary, both CXCL9 peptides inhibited neutrophil migration in vivo through interference with GAG interactions in several animal models. Shortening CXCL9(74-103) from the COOH-terminus limited its GAG-binding spectrum.

Glycosaminoglycans are important mediators of neutrophilic inflammation in vivo.

The pro-inflammatory chemokine interleukin-8 (CXCL8) exerts its function by establishing a chemotactic gradient in infected or damaged tissues to guide neutrophil granulocytes to the site of inflammation via its G protein-coupled receptors (GPCRs) CXCR1 and CXCR2 located on neutrophils. Endothelial glycosaminoglycans (GAGs) have been proposed to support the chemotactic gradient formation and thus the inflammatory response by presenting the chemokine to approaching leukocytes. In this study, we show that neutrophil transmigration in vitro can be reduced by adding soluble GAGs and that this process is specific with respect to the nature of the glycan. To further investigate the GAG influence on neutrophil migration, we have used an engineered CXCL8 mutant protein (termed PA401) which exhibits a much higher affinity towards GAGs and an impaired GPCR activity. This dominant-negative mutant chemokine showed anti-inflammatory activity in various animal models of neutrophil-driven inflammation, i.e. in urinary tract infection, bleomycin-induced lung fibrosis, and experimental autoimmune uveitis. In all cases, treatment with PA401 resulted in a strong reduction of transmigrated inflammatory cells which became evident from histology sections and bronchoalveolar lavage. Since our CXCL8-based decoy targets GAGs and not GPCRs, our results show for the first time the crucial involvement of this glycan class in CXCL8/neutrophil-mediated inflammation and will thus pave the way to novel approaches of anti-inflammatory treatment.

Profiling the Membrane and Glycosaminoglycan-Binding Proteomes of Moraxella catarrhalis.

Moraxella catarrhalis, a Gram-negative bacterium, is an important respiratory pathogen causing acute otitis media and exacerbations of chronic obstructive pulmonary disease. Adhesion of the pathogen to human epithelial cells is mediated via bacterial membrane adhesin proteins. To identify the surface proteome of Moraxella catarrhalis, we applied different membrane protein extraction methods in combination with different proteomic technologies. Proteins from preparations of outer membrane vesicles and from carbonate extractions were analyzed using either a gel-based nano-HPLC-MS/MS technique or 2D-LC-MS/MS. Furthermore, because glycosaminoglycans (GAGs) play an important role for microbial entry into human cells, the GAG-binding membranome of Moraxella catarrhalis was investigated using a glycan-based pull-down approach. By these means, potential vaccine protein candidates that were previously selected by the ANTIGENome technology were confirmed, but importantly also novel proteins were identified as candidates.

Interfering with the CCL2-glycosaminoglycan axis as a potential approach to modulate neuroinflammation.

Multiple Sclerosis, a chronic inflammatory demyelinating disease of the central nervous system, involves an increased expression of monocyte chemotactic protein 1 MCP1-/CCL2. For exerting its chemotactic effects, chemokine binding to glycosaminoglycans (GAGs) is required and therefore this interaction represents a potential target for therapeutic intervention. We have designed an anti-inflammatory decoy variant, Met-CCL2 (Y13A S21K Q23R), embodying increased affinity for GAGs as well as knocked-out GPCR activation properties. This non-signalling dominant-negative mutant is shown here to be able to displace wild type CCL2 from GAGs by which it is supposed to interfere with the chemokine-related inflammatory response. In vivo, the anti-inflammatory properties were successfully demonstrated in a murine model of zymosan-induced peritonitis as well as in an experimental autoimmune encephalomyelitis, a model relevant for multiple sclerosis, where the compound lead to significantly reduced clinical scores due to reduction of cellular infiltrates and demyelination in spinal cord and cerebellum. These findings indicate a promising potential for future therapeutic development.

Exploring the glycosaminoglycan-protein interaction network by glycan-mediated pull-down proteomics.

Glycosaminoglycans (GAGs) are linear, highly sulfated polysaccharides expressed by almost all animal cells. They occur as soluble molecules, or form proteoglycans by being O-linked to different core proteins on the cell surface and in the extracellular matrix. Due to their ability to interact with diverse proteins and to modulate their biologic functions, GAGs are main drivers of mammalian biology. However, to the present day, the human GAG binding proteome has only been insufficiently explored. The aim of this study was therefore to investigate the human GAG binding proteome of different sources by using the major GAG classes as ligands, and to explore the GAG-binding selectivity of the human plasma proteome. For this purpose, proteins were pulled down from immobilized low molecular weight heparin, heparan sulfate, and dermatan sulfate under different conditions and were identified by nano-LC/MS². Four hundred and fifty eight human GAG binding proteins have been identified, whereas plasma proteins showed clear differences in their GAG-binding specificity/selectivity and affinity. We were able to differentiate between proteins that bound to all three glycan ligands and proteins that showed selective binding to one or two glycan ligands. Moreover, step-gradient salt elution revealed different binding affinities toward different GAG ligands. On top of proteins with well-known GAG-binding properties we have identified formerly unknown GAG binders. Functional annotation of the identified GAG-binding proteins showed clusters of proteins that are involved in a variety of biological processes. The method described here is well suited for identifying GAG-binding proteins and for comparing human subproteomes with respect to binding to different GAG classes.